The hedgehog genes encode inductive factors which regulate diverse aspects of vertebrate and invertebrate development. Analysis of these signals has led to critical insights into the basis of embryonic induction and morphogenesis. One of the most important model systems for understanding organogenesis is the development of the vertebrate limb. Sonic hedgehog (Shh) is the key morphogenic signal produced in the posterior limb bud which organizes the anterior-posterior axis of the developing limb bud. While much has been learned in the last decade concerning the mechanisms of hedgehog signal transduction, how this is translated into instructions for pattern and form is still poorly understood. This will be the broad topic of our work in the next funding period, with specific focus on four related issues. First, we have recently found evidence that differential length of time of exposure to Shh is critical in patterning the posterior limb bud in addition to concentration of Shh. This model will be confirmed in experiments testing the effect of dose and time of Shh expression, by blocking Shh activity at various stages during normal chick limb development, and truncating the time of Shh exposure genetically in mouse limb buds. Second, we will set up an in vitro limb culture system where we can test the response of specific target genes of the Hoxd complex to varying concentrations and times of exposure to Shh and can correlate these with changes in processing and expression of GN3, the key downstream transcriptional regulator in Shh signal transduction in the limb. Third, we will investigate whether skeletal, tendon, and/or muscle progenitors are directly patterned by Shh signaling by conditionally removing activity of the Shh effector Smo in mouse cells of each of these lineages. Fourth, we will conduct a functional screen in the developing chick limb bud to identify transcription factors activated by Shh signaling, which serve as a molecular memory of early Shh patterning during later stages of organogenesis in the limb. [unreadable] [unreadable]